CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Corresponding author: Robert M. Stupar

Processing of double-stranded RNA precursors (前体) into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient (不足的) mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean (大豆) and Medicago truncatula (苜蓿) were generated using the CRISPR/Cas9 and TAL-effector nuclease (TALEN; 类转录激活因子效应物核酸酶) mutagenesis (诱变) platforms. An efficient CRISPR/Cas9 reagent (试剂) was used to create a bi-allelic double mutant (双等位基因双突变体) for the two soya bean paralogous Double-stranded RNA-binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9-generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ-line (生殖系) transmissible (可遗传的). Furthermore, TALENs were used to generate a mutation within the soya bean Dicer-like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer-like3 gene and the GmHen1a gene was observed in the T0 generation, but these mutations failed to transmit to the T1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole-genome sequencing to reveal a spectrum (谱) of non-germ-line-targeted mutations and multiple transgene insertion events. Finally, a suite (一套) of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic (协作的) use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.

双链RNA前体加工为小RNA对于植物发育和胁迫响应中基因表达的调控是至关重要的。小RNA的处理过程需要不同功能化的分子组分共同作用。然而，在大多数植物物种中，并没有足够的突变体资源对每个编码基因进行功能研究。本文利用CRISPR/Cas9和类转录激活因子效应物核酸酶TALEN诱变平台获得了在大豆和苜蓿中参与小RNA处理过程的蛋白编码基因位点的突变体。作者利用一个有效的CRISPR/Cas9系统对大豆两个旁系同源基因GmDrb2a和GmDrb2b进行了双等位基因双突变。这些突变体和利用CRISPR/Cas9获得的苜蓿MtHen1基因突变体在生殖系中被证明可遗传。此外，作者利用TALEN获得了大豆Dicer-like2基因的突变体。大豆Dicer-like3和GmHen1a基因的CRISPR/Cas9诱变在T0代中可观测到，然而这些突变并不能够传递到T1代。作者通过全基因组测序对这些诱导突变的不规则遗传和相应的转基因基因了深入研究，揭示了一系列的非生殖系靶向的突变和多个转基因插入事件。最终，结合先前报道的Gmdcl1a，Gmdcl1b和Gmdcl4b突变体及Gmdrb2ab双突变体，作者获得了一套组合的突变体植株。综上，本文揭示了利用不同基因组工程平台的协作，可以产生一系列有用的突变体植物株系，用以未来在豆类作物中的小RNA加工研究。

通讯：Robert M. Stupar (https://agronomy.cfans.umn.edu/department-directory/robert-m-stupar)

个人简介：2005年，威斯康星州，博士。

doi: 10.1111/pbi.12857